Multifunctional hyaluronic acid-based biomimetic/pH-responsive hybrid nanostructured lipid carriers for treating bacterial sepsis

Abstract

Introduction: The application of biomimetic and stimuli-responsive nanocarriers displays considerable promise in improving the management of bacterial sepsis and overcoming antimicrobial resistance. Therefore, the study aimed to synthesize a novel hyaluronic acid-lysine conjugate (HA-Lys) and to utilize the attributes of the synthesized HA-Lys with Tocopherol succinate (TS) and Oleylamine (OLA) in the formulation of multifunctional biomimetic pH-responsive HNLCs loaded with vancomycin (VCM-HNLCs), to combat bacterial sepsis.

Methods: A novel hyaluronic acid-lysine conjugate (HA-Lys) was synthesized and characterized using FTIR and ¹H NMR spectroscopy. Vancomycin-loaded hybrid nanosystems (VCM-HNLCs) were prepared through hot homogenization ultrasonication and evaluated for particle size, polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE%). In vitro biocompatibility was assessed via MTT assay and red blood cell hemolysis test. The binding affinity to TLR2 and TLR4 was measured using microscale thermophoresis (MST). Drug release was evaluated using the dialysis bag method. Antimicrobial activity against MRSA and efflux pump inhibition were also determined. Efficacy was demonstrated in an MRSA-induced sepsis mice model.

Results: The VCM-HNLCs, produced via hot homogenization ultrasonication, exhibited particle size (PS), polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE%) of 110.77 ± 1.692 nm, 0.113 ± 0.022, - 2.92 ± 0.210 mV, and 76.27 ± 1.200%, respectively. In vitro, biocompatibility was proven by hemolysis and cytotoxicity studies. The VCM-HNLCs demonstrated targetability to human Toll-like receptors (TLR 2 and 4) as validated by microscale thermophoresis (MST). VCM-HNLCs showed a twofold reduction in MIC values at physiological pH compared to the bare VCM against S. aureus and MRSA for 48 h. While at pH 6.0, MIC values were reduced by fourfold in the first 24 h and by eightfold in the subsequent 48 and 72 h against tested strains. Furthermore, VCM-HNLCs showed inhibitory effects against MRSA efflux pumps, reactive oxygen species (ROS), and lipopolysaccharide (LPS)-induced hyperinflammation. In an MRSA-induced sepsis mice model, VCM-HNLCs demonstrated superior efficacy compared to free VCM, significantly eliminated bacteria and improved survival rates.

Conclusions: Overall, these results highlight the potential of VCM-HNLCs as novel multifunctional nanocarriers to combat antimicrobial resistance (AMR) and enhance sepsis outcomes.

Keywords: Antibiotic delivery; Bacterial sepsis; Biomimetic; Hyaluronic acid-lysine; Hybrid nanocarriers; PH-responsive.

Scheme 1.

Synthesis of HA-Lys conjugate using a one-step amidation reaction. (EDC: 1-ethyl-3- (dimethyl aminopropyl) carbodiimide, NHS: N-hydroxy succinimide)

Fig. 1

The impact of pH variation on the surface charge of VCM-HNLCs (P-value = 0.0004). The data is shown as mean ± SD (n = 3)

Fig. 2

A TEM image of VCM-HNLCs and B A histogram displaying the size distribution of VCM-HNLCs determined by DLS

Fig. 3

The cell viability % of different concentrations of A HA-Lys conjugate and B VCM-HNLCs, and controls on HEK293 and HepG2 cell lines. Data are presented as mean ± SD (n = 3)

Fig. 4

A, B Non-hemolytic activity of different concentrations of VCM-HNLCs (*DW = distilled water, PBS = phosphate buffer saline pH 7.4). Data are presented as mean ± SD (n = 3)

Fig. 5

A Comparison view for different ligands fraction bound to A TLR2 and B TLR4. PGN: Peptidoglycan, LPS: lipopolysaccharide, HA: hyaluronic acid, HA-Lys: hyaluronic acid-lysine conjugate and VCM-HNLCs: vancomycin-loaded hybrid nanostructured lipid carriers. Results represented in mean ± SD (n = 3)

Fig. 6

Drug release profile of VCM-HNLCs and bare VCM at pH 7.4 and pH 6.0. All data is represented as mean ± SD (n = 3)

Fig. 7

Variations in particle size (nm) and ZP (mV) of VCM-HNLCs during a ninety-day storage at A room temperature (RT) and B 4 °C. All results are presented as mean ± SD (n = 3)

Fig. 8

Histogram of cell count versus propidium iodide (PI) uptake. A corresponds to the untreated MRSA (live cells); B, C and D indicate the proportion of dead MRSA cells following incubation with bare VCM at its MIC (7.8 μg/mL), bare VCM in NPs MIC (3.9 μg/mL) and VCM-HNLCs at its MIC (3.9 μg/mL), respectively. The experiment was conducted in triplicate (n = 3)

Fig. 9

A Percentage of biofilm growth suppression following the exposure of MRSA biofilms to bare VCM, VCM-HNLCs and controls (PBS pH 7.4 and 6.0). Each result is shown as mean ± SD (n = 3) (P-values **** < 0.0001). B MRSA biofilm growth reduction after exposure to bare VCM and VCM-HNLCs at PBS pH 7.4 and 6.0, as indicated by changes in the crystal violet intensity

Fig. 10

Images of the fluorescence microscope assay of MRSA biofilms stained with Syto9 and PI. 1. Untreated MRSA biofilms 2. Bare VCM-treated biofilms, and 3. VCM-HNLCs treated biofilms, respectively. (Scale bar = 100 µm)

Fig. 11

MRSA time-killing kinetics when exposed to bare VCM, VCM-HNLCs and A PBS pH 7.4 and B pH 6.0 (as controls). The findings are shown as mean ± SD (n = 3). At pH 6, VCM-HNLCs demonstrated more enhancement of VCM-killing kinetics (P-value < 0.0001)

Fig. 12

Efflux pump inhibition effect of VCM-HNLCs against MRSA compared to the bare VCM at 0.25 × MIC

Fig. 13

The antioxidant activity (%) of various concentrations (µg/mL) of TS, Lys, HA-Lys conjugate and VCM-HNLCs according to a DPPH radical scavenging assay in comparison to ascorbic acid (positive control). Data in the graph are presented as mean ± SD (n = 3). (TS: Tocopherol succinate, Lys: L-Lysine, HA-Lys: hyaluronic acid-lysine conjugate, VCM-HNLCs: vancomycin-loaded hybrid nanostructured lipid carriers, DPPH: 2.2-diphenyl-1-picrylhydrazyl)

Fig. 14

A The Cytotoxicity of LPS on HepG2 cells and the protective effects of HA-Lys and VCM-HNLCs as measured by MTT assay, and B The effect of HA-Lys and VCM-HNLCs on IL-6 expression induced by LPS. (****P-value < 0.0001, ***P-value = 0.0002, in comparison with the LPS-treated group). Data in the graph are presented as mean ± SD (n = 3)

Fig. 15

Survival and CFUs recovered in different organs in MRSA-induced systemic bacteremia. A Kaplan–Meier survival curves depicting the survival of different treatment groups following MRSA infection. B Comparison of CFUs recovered in blood, liver, kidney, heart, lungs, and spleen after treatment with bare VCM and VCM-HNLCs.